ABSTRACT Phosphoglucosamine mutase (EC 5.4.2.10) catalyzes the interconversion of glucosamine-6-phosphate into glucosamine-1-phosphate, an essential step in the biosynthetic pathway leading to the formation of peptidoglycan precursor uridine 5'-diphospho-N-acetylglucosamine. The gene (glmM) of Escherichia coli encoding the enzyme has been identified previously. We have now identified a glmM homolog in Streptococcus gordonii, an early colonizer on the human tooth and an important cause of infective endocarditis, and have confirmed that the gene encodes phosphoglucosamine mutase by assaying the enzymatic activity of the recombinant GlmM protein. Insertional glmM mutant of S. gordonii did not produce GlmM, and had a growth rate that was approximately half that of the wild type. Morphological analyses clearly indicated that the glmM mutation causes marked elongation of the streptococcal chains, enlargement of bacterial cells, and increased roughness of the bacterial cell surface. Furthermore, the glmM mutation reduces biofilm formation and increases sensitivity to penicillins relative to wild type. All of these phenotypic changes were also observed in a glmM deletion mutant, and were restored by the complementation with plasmid-borne glmM. These results suggest that, in S. gordonii, mutations in glmM appear to influence bacterial cell growth and morphology, biofilm formation, and sensitivity to penicillins.

[Show abstract][Hide abstract]ABSTRACT:
The biosynthetic pathway responsible for the production of hyaluronic acid (HA) has been thoroughly studied; however, many aspects remain elusive regarding the mechanisms that control molecular weight (MW). Previously, we demonstrated a positive correlation between MW and the concentration of the HA precursor sugar UDP-N acetylglucosamine (UDP-GlcNAc). To further investigate the role of UDP-GlcNAc in MW control, we increased the intracellular concentration of this monomer using both feeding strategies and genetic engineering approaches. Feeding cells glucosamine dramatically increased intracellular levels of UDP-GlcNAc, but unexpectedly, produced HA of a lower MW. This was subsequently attributed to an equally dramatic decrease in the level of the other HA precursor sugar UDP-glucuronic acid (UDP-GlcUA). Feeding cells a mixture of glucose and GlcNAc addressed this imbalance of precursor sugars, leading to an increase in both UDP-GlcNAc and UDP-GlcUA; however, no significant increase in MW was observed. Despite the increase in UDP-sugars, RNA sequencing identified no increase in the expression of the genes involved in production of HA. Returning to genetic engineering approaches to examine UDP-GlcNAc and MW, genes known to contribute to the production of UDP-GlcNAc were over-expressed, both individually and together. Using this approach, UDP-GlcNAc and MW increased. At lower levels of UDP-GlcNAc, the positive correlation between UDP-GlcNAc levels and MW was maintained, however this relationship stalled at higher concentrations of UDP-GlcNAc. Taken together, these results suggest that while optimising HA precursor levels using feeding or genetic engineering approaches can improve HA MW, there is a point at which excess availability of precursors is no longer advantageous. Once precursor concentrations are addressed, it would seem that other uncharacterised factor(s) (e.g. rate of HA synthesis) also contribute to HA MW control.

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Bacteria lipopolysaccharides (LPS) contain a great variety of sugars including neutral sugars, sugar acids, amino sugars and different deoxy-sugars. The sugar units must be converted into sugar nucleotides (NDP sugars) before they are recognized by specific glycosyltransferases and assembled into a sugar polysaccharide one by one. We were invited by the editors, Dr. Knirel and Dr. Valvano to write this review, in which we summarized the current knowledge of the biosynthesis pathways of more than 30 of the NDP sugars that has been found in the LPS or surface polysaccharides of bacteria (some of these NDP-sugars are especially important to the virulence of pathogens). We grouped these NDP sugar synthesis pathways according to their original sugar sources (Glc-6-P, Fru-6-P or UDP-D- GlcNAc) and the identity of the coupled NDP (dTDP, GDP or UDP). We also addressed some common rules in NDP-sugar biosynthesis pathways.

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Preparation of His6-tagged GlmM enzymePCR product F201, containing glmM of S.gordonii DL1, wascloned into the expression vector pET200/D-TOPO (Invi-trogen), generating plasmid pIRG201. DNA sequencing wasperformed to confirm that the sequence of the construct wascorrect. The plasmid was then transformed into E. coli BL21Star (DE3) (Invitrogen). His6-tagged recombinant GlmM(i.e. His6-GlmM) enzyme was prepared from the recombi-nant as described previously (Jolly et al., 1997). PurifiedHis6-GlmM was dialyzed against 20mM potassium phos-phate buffer (pH 7.4) containing 0.5mM MgCl2and 0.1%b-mercaptoethanol and stored with 40% (v/v) glycerol at?201C for later use. The purity of the prepared His6-GlmMwas confirmed by sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE) analysis (Laemmli, 1970)using 4–12% Bis-Tris Gel (NuPAGE; Invitrogen), followedby staining with Coomassie Brilliant Blue (Fairbanks et al.,1971). Protein concentrations were determined by themethod of Bradford (Bradford, 1976), using bovine serumalbumin as the standard.Preparation of His6-tagged GlmU enzymeHis6-tagged GlmU (i.e. His6-GlmU) enzyme was preparedas described previously (Pompeo et al., 1998) from therecombinant E. coli BL21 Star (DE3) harboring pIRG202,in which glmU of E. coli W3110 (Jensen, 1993) was cloned.The purity of the prepared His6-GlmU was primarilyconfirmed by SDS-PAGE, followed by dye staining. Theenzymatic activity of His6-GlmU was primarily confirmedby the assay described in the next section, except thatglucosamine-1-phosphate was used as the substrate insteadof glucosamine-6-phosphate (Mengin-Lecreulx & van Hei-jenoort, 1993).Enzymatic assayThe coupled assay in which the glucosamine-1-phosphatesynthesized from glucosamine-6-phosphate by the mutasewas quantitatively converted to UDP-GlcNAc in the pre-sence of purified bifunctional GlmU enzyme, possessingboth glucosamine-1-phosphate acetyltransferase activityand GlcNAc-1-phosphate uridyltransferase activity (Men-gin-Lecreulx & van Heijenoort, 1993, 1994), was performedas described previously (Mengin-Lecreulx & van Heijenoort,1996) with some modifications. The chemicals used in thisassay were purchased from Sigma-Aldrich. The assay mix-ture contained 50mM Tris-HCl buffer (pH 8.0), 3mMMgCl2, glucosamine-6-phosphate (0, 0.5 or 1mM), 0.4mMacetyl coenzyme A, 0.7mM glucose-1,6-diphosphate, 2mMuridine 50-triphosphate (UTP), His6-GlmU (1mg protein),and His6-GlmM (4mg protein) in a final volume of 50mL.The mixture was incubated at 371C for 30min, and thereaction was terminated by heating at 1001C for 5min. Thereaction products were isocratically separated by HPLCusing a reverse-phase column (ODS-80Ts, Tosoh, Tokyo,Japan) with 20mM triethylamine-acetic acid buffer (pH4.0) at a flow rate of 1mLmin?1(LC-9A pumps, ShimazduCo. Ltd, Kyoto, Japan) (Rabina et al., 2001; Wopereis et al.,2006). The A260nmof the eluate was monitored using anultraviolet spectrophotometric detector (SPD-6A, Shimad-zu). UDP-GlcNAc and UTP were eluted at 8.9 and 14min,respectively, after the mixture was applied to the column(Fig. 1). The amount of UDP-GlcNAc produced was calcu-lated by comparing the chromatogram with that of 1mMUDP-GlcNAc as the standard.Insertional mutation, deletion andcomplementation of glmM in S. gordoniiAn erythromycin-resistance gene (ermAM) was insertedinto glmM of S. gordonii DL1 to obtain a glmM::erm mutant(EM231, supplementary Fig. S1). Initially, the ermAMfragment cleaved from pMDC10E (Shiroza et al., 1998) wasinserted at the SspI site of the F001 fragment in plasmidpIRG001. The resulting plasmid, pIRE001, was linearized bydigestion with HindIII and XbaI, and was transformed intoS. gordonii DL1. The transformants thus produced weregrown in the presence of erythromycin to select for recom-bination between the regions flanking ermAM and homo-logous regions in glmM. Insertion of ermAM into theexpected locations in the transformant (i.e. EM231) wasverified by Southern blotting of chromosomal DNA usingthe ermAM gene fragment as a probe and by PCR withprimers flanking the predicted sites of ermAM insertion(primer pair for F001, Table S2).The S. gordonii DL1 chromosomal DNA fragment con-taining glmM was replaced with a chloramphenicol-resis-tance gene (cat) to obtain DglmM mutant (CM201,supplementary Fig. S1) for use in genetic complementationstudies. DNA fragments (c. 500bp each) on either side ofS. gordonii DL1 glmM, F201UP and F201DN (supplemen-tary Table S2), were amplified by PCR, digested with theappropriate restriction endonucleases, and inserted eitherside of the cat gene into plasmid pR326 (Claverys et al.,1995). The resulting plasmid (pIRC201) was linearized bydigestion with BglII and SphI, transformed into S. gordoniiDL1, and a single transformant (CM201) was selectedfollowing growth on chloramphenicol. The replacement ofglmM by cat was verified by Southern blotting of CM201chromosomal DNAusing the cat and glmM genes as probes,and by PCR with forward primer for F201UP and reverseprimer for F201DN respectively, or a primer pair for cat.Genetic complementation of S. gordonii CM201 with theglmM gene was performed by transformation with pAS201(supplementary Fig. S1), a streptococcal plasmid containingFEMS Immunol Med Microbiol 53 (2008) 166–177c ?2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved168K. Shimazu et al.

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the glmM gene and its putative promoter region. PCR-amplified F201C was inserted into pAS41S (supplementaryTable S1), which was subsequently transformed intoS. gordonii CM201. Spectinomycin-resistant (Spr) transfor-mant was isolated as S. gordonii CM201(pAS201) (DglmM/glmM). Plasmids were isolated from the transformants andanalyzed by digestion with appropriate restriction endonu-cleases to confirm the presence of the expected insert.Immunological proceduresRabbit antiserum against GlmM (anti-GlmM) was preparedbyimmunizingrabbitsproteininjection?1) emulsified with an equal volume ofFreund’s complete adjuvant (Sigma-Aldrich) and adminis-tered subcutaneously at multiple sites. Anti-GlmM serumwas obtained 1 week after three subsequent injections of theantigen in an incomplete adjuvant administered at biweeklyintervals.Sonic extracts of streptococci were prepared for Westernblot analysis as described previously (Takahashi et al., 1997),separated by SDS-PAGE as described above, and electro-phoretically transferred to nitrocellulose membranes (Tow-bin et al., 1979). The membranes were blocked with Tris-buffered saline (TBS) containing 1% bovine serum albumin,incubated with anti-GlmM (1:100 dilution), washedwith TBS containing 0.05% Tween 20, and incubatedwith peroxidase-conjugated goat anti-rabbit IgG (1:1000dilution; Bio-Rad, Hercules, CA). Bound antibody wasdetected using 4-chloro-1-naphthol and H2O2.with His6-GlmM (0.3mgBacterial growthBHI broth (5mL) in a glass tube was inoculated with an18–24-h streptococcal culture (final A620nm=0.01). Bacter-ial cell growth in a static culture at 371C was automaticallyrecorded at A660nmusing the TVS062CA Bio-photorecorder(Advantec, Tokyo, Japan).Biofilm assaysAn assay measuring biofilm formation on a glass wall wasperformed as described previously (Olson et al., 1972) withsome modifications. BHI broth supplemented with 5%sucrose (3mL) in a glass tube was inoculated with an18–24-h streptococcal culture (final A620nm=0.01). Thetube was incubated at 371C for 20h while statically posi-tioned at an angle of c. 151 from the horizontal to increasethe surface area for biofilm formation. Bacteria nonadheredto the glass wall of the tube were taken off with the mediainto another glass tube. Adhered bacteria remaining on theglass wall were suspended in 3mL of TBS (20mM Tris-HCl[pH 7.8], 150mM NaCl) by vigorous shaking. The A620nmof each suspension was measured. Percentage adherence wasdefined as (A620nmof adhered cell suspension)/[(A620nmof adhered cell suspension)1(A620nmof nonadhered cellsuspension)]?100.Fig. 1. HPLC chromatogramfor measurement of the amount of productin the coupled assay in which the glucosamine-1-phosphate synthesizedfrom glucosamine-6-phosphate (GlcN-6-P) by phosphoglucosamine mu-tase (i.e. GlmM) was quantitatively converted into UDP-GlcNAc in thepresenceof purified bifunctional GlmU enzyme and its substrates, acetyl-coenzyme A and UTP. The standards contained 1mM UDP-GlcNAc and0.5mM UTP. UDP-GlcNAc and UTP, which eluted at 8.9 and 14min,respectively, are indicated by arrow heads.FEMS Immunol Med Microbiol 53 (2008) 166–177c ?2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved169Streptococcus gordonii phosphoglucosamine mutase

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Biofilm formation on plastic walls in culture with mixingwas also assessed using the MBECTMPhysiology and Genet-ics assay of the Calgary Biofilm Device (Innovotech Inc.,Edmonton, Canada) as described previously (Bernier &Sokol, 2005) with some modifications. The sucrose-supple-mented BHI broth inoculated with an 18–24-h streptococcalculture (final A620nm=0.01) was added to each well(150mLwell?1) of the 96-well tissue culture plate of thedevice, and the lid was vertically studded with the 96polystyrene pegs corresponding to the 96 wells, primarilyimmersed in 10% horse serum in phosphate-buffered saline(pH 7.4) for 1h, was placed. The plate was incubated at371Cfor 20hwhilebeing mixed using agyrorotary shaker atc. 100r.p.m. The pegs studded to the lid were immersed in1% crystal violet in a 96-well microtiter plate (200mLwell?1)for 15min, rinsed briefly twice with saline, and immersed inabsolute ethanol in a 96-well microtiter plate (200mLwell?1)for 15min to dissolve the dye. The A575nm of the dyesolution (Biofilm Abs) was measured. The culture mediumcontaining planktonic cells was transferred to another 96-well microtiter plate, and the A575nmof the suspension(Planktonic Abs) was measured. The biofilm formationindex was defined as the ratio of Biofilm Abs/PlanktonicAbs.Morphological methodsBacterial cells cultivated in BHI broth at 371C for 20h wereobserved by Gram staining (Hucker’s modification).Bacterial cells were further observed by transmissionelectron microscopy (TEM). Bacterial pellets were fixedovernight with 2.5% glutaraldehyde and 0.05% rutheniumred in 0.1M cacodylate buffer (pH 7.4) at 41C. Cells werewashed in the same buffer and postfixed for 3h in 1%osmium tetroxide containing 0.05% ruthenium red at 41C.Subsequently, the cells were rinsed with the same bufferagain. Bacteria were suspended for 1h at room temperaturein 2% aqueous uranyl acetate and then washed in distilledwater. Suspended cells were embedded in 1.5% agarosebefore dehydration with a graded ethanol series. The dehy-drated blocks were embedded in Spurr resin. Ultrathinsections were cut with an ultramicrotome (Ultracut-N;Reichert-Nissei, Tokyo, Japan), mounted on copper grids,and stained with uranyl acetate and lead citrate. Images ofsections were obtained using a transmission electron micro-scope (75kV; H-7000; Hitachi, Tokyo, Japan).Determination of minimum inhibitoryconcentrations (MICs) and minimumbactericidal concentrations (MBCs)MICs and MBCs of various antibiotics were determinedusing the microdilution method as described elsewhere(Baker et al., 1981) with BHI broth as medium. In addition,MICs and minimum biofilm eradication concentration(MBECs) in biofilms of S. gordonii strains were determinedusing the MBECTMHigh-throughput assay of the CalgaryBiofilm Device (Innovotech) as described previously (Ceriet al., 1999; Kostenko et al., 2007), except that pegs of thedevice were coated with horse serum before the biofilmformation and the sucrose-supplemented BHI broth wasused as medium. All antibiotics were purchased fromSigma-Aldrich.Statistical analysisStatistical differences in the means of obtained values wereevaluated by an unpaired t-test. Differences were consideredto be significant at Po0.05.Nucleotide sequence accession numberThe DDBJ/EMBL/GenBank accession number assigned tothe glmM gene of S. gordonii DL1 is AB330077.ResultsCloning and DNA sequencing analysis of theS. gordonii DL1 glmM geneA BLAST search for the glmM gene of E. coli against the S.gordonii NCTC7868 (Challis) genome database (the genomeis currently being sequenced at the Institute for GenomicResearch, http://www.tigr.org/) revealed a putative geneencoding a protein that was 43% identical to GlmM of E.coli (Mengin-Lecreulx & van Heijenoort, 1996). The glmMhomolog of S. gordonii DL1 was cloned into E. coli plasmidpZErO-2 (i.e. pIRG101), and the DNA was sequenced. TheglmM gene of S. gordonii consists of 1350 nucleotides andencodes a polypeptide (GlmM) of 450 amino acid residueswith a molecular weight of 48365. The gene is preceded by aputative prokaryotic promoter and a ribosome-binding site,and is followed by a stem-and-loop structure. The aminoacid sequence of GlmM was compared with GenBanksequences using a BLASTP search; GlmM of S. gordonii DL1was found to be completely identical to that of S. gordoniiNCTC7868, and 59%, 42%, and 39% identical to GlmM inS. aureus (Jolly et al., 1997), P. aeruginosa (Tavares et al.,2000), and H. pylori (De Reuse et al., 1997), respectively.Two serine residues that form the active site (Jolly et al.,1999) are conserved in GlmM in S. gordonii DL1 and allother bacteria (Fig. 2).Overexpression and enzymatic activity of His6-tagged recombinant GlmM of S. gordoniiHis6-tagged recombinant protein encoded by glmM ofS. gordonii DL1 (i.e. His6-GlmM) was overexpressed inE. coli BL21 Star (DE3). The His6-GlmM expressed wasFEMS Immunol Med Microbiol 53 (2008) 166–177c ?2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved170K. Shimazu et al.

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confirmed by SDS-PAGE to be a 50-kDa protein (Fig. 3a),which was consistent with the value calculated on the basisof the DNA sequence. The specific activity of purified His6-GlmM was 0.19mmolmin?1mg?1(Fig. 1), approximatelytwice that of the corresponding enzyme in S. aureus (Jollyet al., 1997), and one-tenth that in E. coli (Mengin-Lecreulx& van Heijenoort, 1996) and P. aeruginosa (Tavares et al.,2000). These results confirm that glmM in S. gordonii DL1encodes phosphoglucosamine mutase.Insertional mutation, deletion andcomplementation of the glmM gene inS. gordoniiAn insertional mutant (glmM::erm) was prepared for S.gordonii DL1 (EM231, supplementary Fig. S1) to assess theinvolvement of glmM in biofilm formation, sensitivity toantibiotics, and bacterial cell morphology in this organism.That the glmM::erm mutant, in which ermAM was insertedat a position corresponding to nucleotide 1123 of glmM, didnot produce GlmM was confirmed by Western blotting ofsonicated cell extracts (Fig. 3b). Purified His6-GlmM andsonicated extracts of wild-type DL1 cells served as positivecontrols in the experiment. GlmM production of DglmMmutant (CM201, supplementary Fig. S1) was also abolished,but the production was restored by plasmid-borne glmMcomplementation (DglmM/glmM) (Fig. 3b).Mutation of the glmM gene reduces bacterialgrowthCertain genes encoding enzymes involved in bacterial cellwall synthesis have been associated with bacterial growthand cell separation (Kajimura et al., 2005), (Komatsuzawaet al., 2004). These findings prompted us to examine thepossible role of GlmM in bacterial growth. Compared withthe wild-type strain, the growth rates of the glmM::ermmutant and the DglmM mutant were reduced, as assessed bycultivating the strains statically overnight in BHI broth andthe growth was monitored on the basis of the turbidity ofthe broth culture (Fig. 4a). The growth rates of DglmMFig. 2. Alignment of the predicted amino acid sequence of the activesite of GlmM from Streptococcus gordonii DL1, Escherichia coli, Helico-bacter Pylori, and Staphylococcus aureus. The conserved serine residuesare indicated in bold and with arrows.Fig. 3. (a) Preparation of the recombinant phosphoglucosamine mutase(His6-GlmM). A sonic extract of Escherichia coli BL21 Star (DE3) harbor-ing plasmid pIRG201 was clarified by ultracentrifugation (crude; 20mgprotein) and purified His6-GlmM (His6-GlmM, 2mg protein) were ana-lyzed by SDS-PAGE with molecular mass marker (marker) followed bystaining with Coomassie Brilliant Blue. The positions of the molecularmass markers are indicated on the left. (b) Western blot of streptococcalsonic extracts (50mgproteinlane?1) and purified His6-GlmM (0.5mgprotein) with anti-GlmM (1:100 dilution). The positions of the molecularmass markers are indicated on the left. The positions of a nonspecificantigen (?), His6-GlmM and GlmM are indicated on the right.Fig. 4. Bacterial growth of Streptococcus gordonii in BHI broth. (a)Growth curve. (b) Overnight cultures in glass tubes.FEMS Immunol Med Microbiol 53 (2008) 166–177c ?2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved171Streptococcus gordonii phosphoglucosamine mutase

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mutant were restored in the DglmM/glmM strain (Fig. 4a).Similar results were obtained when growth was monitoredon the basis of CFU, bacterial total protein, and bacterial wetweight (data not shown). Interestingly, most bacterialcells of the glmM mutants sank, but did not adhere,to the bottom of the tube, whereas those of wild-type andDglmM/glmM strainsgrew(Fig. 4b).dispersedinthe brothMutation of the glmM gene reduces biofilmformationIt has been reported that a transposon-insertional glmMmutant of S. gordonii reduces biofilm formation in staticculture (Loo et al., 2000). To further investigate the role ofFig. 5. Biofilm formation by Streptococcus gordonii in BHI broth con-taining 5% sucrose. (a) Adhesion of streptococcal cells to the glasssurface in static cultures. Mean and SD (n=5) of percent adherence areindicated. (b) Adhesion of streptococcal cells to the polystyrene surfacein mixed cultures. Mean and SD (n=6) of biofilm formation index areindicated.Fig. 6. Morphological analysis of Gram-stained Streptococcus gordoniicells. (a) Light micrographs of Gram-stained streptococci. Scale bar,10mm. (b) Lengths of the streptococcal chain. Mean and SD (n=30) ofbacterial cell number per streptococcal chain are indicated.FEMS Immunol Med Microbiol 53 (2008) 166–177c ?2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved172K. Shimazu et al.

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GlmM, biofilm formation by glmM mutants in static andmixed cultures was compared with that by the wild-typestrain. As shown in Fig. 5, the glmM mutants resulted in asignificant reduction in biofilm formation on the glasssurface in static cultures and on the polystyrene surface ofthe Calgary Biofilm Device in mixed cultures, when thebacteria were grown in BHI broth containing 5% sucrose,the substrate of glucosyltransferase for production of glu-can. These biofilm formations of the DglmM mutant wererestored in the DglmM/glmM strain.The glmM gene has a role in determiningbacterial cell morphologyGiven that GlmM is involved in bacterial cell wall synthesis,it must also play an important role in bacterial cell mor-phology. We found that bacterial chains in a static culture ofthe glmM::erm mutant were markedly elongated relative towild type (Fig. 6a). The chains appeared to be entangledwith other chains. Mutation of glmM, however, seemed notto affect the Gram-staining character of the cells. Theaverage number of bacterial cells per chain was 22 for theglmM::erm mutant, c. 10-fold the number in the wild-typeculture (Fig. 6b). Similar elongation of bacterial chains wasobserved in the DglmM mutant. However, the length of thechains observed in DglmM/glmM was the same as that of thewild type (Fig. 6).Furthermore, TEM revealed variations in cell size andshape in the glmM::erm mutant culture relative to the wildtype. As shown in Fig. 7, most mutant cells were larger, andwith a rougher cell surface than the wild-type cells. Inaddition, variations in cell size and shape were found in themutant cells.Mutation of the glmM gene increases sensitivityto antibioticsThat the glmM mutation in S. aureus results in reducedmethicillin resistance has been reported (Jolly et al., 1997;Glanzmann et al., 1999). To investigate whether glmMmutations in S. gordonii also affect sensitivity to antibiotics,we examined the MICs and MBCs of antibiotics includingpenicillins and inhibitors of protein synthesis with respect totheir effect on S. gordonii cells. As shown in Table 1, theglmM mutants were c. 10-fold more sensitive to penicillinsthan the wild-type or the DglmM/glmM strain. However, allstrains were similarly sensitive to inhibitors of proteinsynthesis. No clear differences of antimicrobial susceptibilitywere found between planktonic bacteria and in the biofilmof each strain.DiscussionIn the present study, the glmM gene of S. gordonii DL1 wascloned on the basis of amino acid sequence similarity to thehomologous enzyme in other bacteria. We confirmed theenzymatic activity of the recombinant GlmM, showing thatglmM encodes phosphoglucosamine mutase. In the enzymeassay, isocratic elution of UDP-GlcNAc and UTP by reverse-phase HPLC allowed us to assess the phosphoglucosaminemutase activity without using radiolabeled reagents. Theactivity of S. gordonii GlmM is quantitatively similar to thatof the GlmM of S. aureus (Jolly et al., 1997), but is c. 10-foldFig. 7. Transmission electron micrographs ofthin-sectioned Streptococcus gordonii cells.Scale bars in the upper panels (high magnifica-tion), and the lower panels (low magnification)represent 200 and 500nm, respectively.FEMS Immunol Med Microbiol 53 (2008) 166–177c ?2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved173Streptococcus gordonii phosphoglucosamine mutase

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less than that of GlmM from Gram-negative bacteria suchas E. coli and P. aeruginosa (Mengin-Lecreulx & vanHeijenoort, 1996).After the glmM gene of E. coli JM83 was identified(Mengin-Lecreulx & van Heijenoort, 1996), homologs in S.aureus, H. pylori and P. aeruginosa were found subsequently(De Reuse et al., 1997; Jolly et al., 1997; Tavares et al., 2000).The present study is the first report in which a streptococcalglmM has been identified and characterized. Using a biofilmassay involving static cultures in 96-well microtiter plates,Loo et al. (2000) found that a glmM transposon mutant ofS. gordonii loses the ability to form biofilms in the normalway. In the present study, we clearly demonstrated that theglmM gene is involved in biofilm formation in both staticand mixed cultures of S. gordonii DL1 by comparison ofwild-type and glmM mutant cells.In S. aureus, certain factors associated with peptidoglycanmetabolism influence resistance to methicillin and vanco-mycin (Berger-Bachi et al., 1989; Maidhof et al., 1991; Henzeet al., 1993; Gustafson et al., 1994; Pinho et al., 2001;Komatsuzawa et al., 2004). Similarly, in the present study,we found that in S. gordonii DL1 mutation of glmM affectssensitivity to penicillins. However, no clear differences ofantimicrobial susceptibility were found between planktonicbacteria and in the biofilm of each strain. These findingssuggest that impaired peptidoglycan synthesis in the glmMmutant leads to an increase in sensitivity to cell wallinhibitors.Our morphological analyses showed that the glmMmutation causes marked elongation of the streptococcalchains in S. gordonii (Fig. 6), suggesting that the mutationmay affect bacterial cell division. The unusual long chainformation may explain why the mutant cells were found atthe bottoms of the culture tubes (Fig. 4b). Further morpho-logical analysis using electron microscopy revealed thatthe mutant cells were larger, with a rougher cell surface thanthe wild-type cells (Fig. 7), suggesting that unusualpeptidoglycan synthesis occurs in the mutant. Furthermore,variations in cell size and shape were found in mutant cells.These morphological findings resemble those observedfor S. aureus with a mutation in sle1, the gene encodingN-acetylmuramyl-L-amidase (Kajimura et al., 2005), sug-gesting that mutations in genes encoding factors involved inpeptidoglycan metabolism influence cell shape, size, anddivision in Gram-positive bacteria.The glmM mutation results in malfunction of glucosa-mine-1-phosphate synthesis. The glmM gene is essential forthe growth of E. coli (Mengin-Lecreulx & van Heijenoort,1996); however, in S. gordonii, although the growth of glmMmutants is reduced, cells are still viable, suggesting that anTable 1. MIC and MBC/MBEC of antibiotics in planktonic and in biofilm of Streptococcus gordonii strainsS. gordonii strainMIC (mgmL?1) in planktonic (upper) and in biofilm (lower)PenicillinsInhibitors of protein synthesisAmpicillinMethicillin Penicillin GSpectinomycin Kanamycin ChloramphenicolWild type 0.250.500.0310.0630.0630.130.250.500.0630.0630.00780.00780.0160.0310.0630.0630.00780.00780.000980.00200.00200.00200.00780.0078100100100100ND252512.512.5252525253.13.11.61.6NDglmM::ermDglmMDglmM/glmMND NDS. gordonii strainMBC (mgmL?1) in planktonic (upper) and MBEC (mgmL?1) in biofilm (lower)Penicillins Inhibitors of protein synthesisAmpicillin MethicillinPenicillin G SpectinomycinKanamycin ChloramphenicolWild type880.50.50.50.522880.130.130.130.13220.510.0160.0160.0310.0310.250.25100100200200ND10010050505050100100100100100100NDglmM::ermDglmMDglmM/glmMNDNDND, not done.FEMS Immunol Med Microbiol 53 (2008) 166–177c ?2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved174K. Shimazu et al.